Q tifi ti f i fl t t ki i Quantification of inflammatory cytokines in dried

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Q tifi ti f i fl t t ki i Quantification of inflammatory cytokines in dried
Quantification
Q
tifi ti off inflammatory
i fl
t
cytokines
t ki
in
i
dried blood spot (DBS) samples
Thom McDade, PhD
Department of Anthropology
Laboratory for Human Biology Research
Cells to Society (C2S):
The Center on Social Disparities and Health
Health,
Institute for Policy Research
Why inflammatory cytokines?



Pathophysiology of
diseases of aging
Sensitive to social and
developmental contexts
Beyond
y
CRP: upstream
p
regulation
g
of inflammation

Pro- and anti-inflammatory inputs

Cytokines cross the blood-brain
blood brain barrier
Measuring inflammation in DBS
CRP
• Low
L
cost,
t robust
b t assay1
• Widespread application
IL-6
• Low concentrations in unstimulated
samples is a major challenge
• Recently validated ELISA2
1McDade
2Miller
et al
al. (2004) Clin Chem 50:652-4.
50:652 4
and McDade (2012) Am J Hum Bio 24:863-5.
Highly sensitive IL-6 ELISA for DBS
Protocol highlights
Assay performance
Adv/disadv

Platform: R&D Systems Quantikine HS IL-6 ELISA

Calibrators: DBS material manufactured in lab with
serum calibrators provided in kit

Sample quantity: 4 x 3.2 mm discs per well
 8 discs for duplicate measures

Samples and calibrators eluted in filter plates to
maximize recovery
Highly sensitive IL-6 ELISA for DBS
Protocol highlights
Assay performance
Adv/disadv

Lower limit of detection: 0.67 pg/mL

Linearity of dilution: 0.80-1.25,
0 80 1 25 mean = 1
1.05
05

Variability
high
med
low
Intra-assay (%CV)
6.3
8.9
14.6
Inter assay (%CV)
Inter-assay
94
9.4
95
9.5
15 4
15.4
Highly sensitive IL-6 ELISA for DBS
Protocol highlights
N=46
Assay performance
Adv/disadv
Highly sensitive IL-6 ELISA for DBS
Protocol highlights
Assay performance
Adv/disadv
Advantages

Tool for measuring IL
IL-6
6 at very low concentrations

Relies on gold-standard R&D protocol

Can be implemented with standard EIA equipment
Disadvantages

R
Requires
i
1
1-2
2d
drops off whole
h l bl
blood
d (f
(for d
duplicates)
li t )

Limited assay range (<10 pg/mL)

Expensive, particularly in comparison with CRP
Advantages
g of multiplexing
p
g

Assay multiple inflammatory cytokines at once
 Pro- and anti-inflammatory signaling
 Patterns of cytokine response (e.g., Th1 vs. Th2)

Conserves sample
 Analyze up to 10 biomarkers in single volume of sample

Lower cost per biomarker (but higher overall cost)
Multiplex analysis of inflammatory
cytokines in DBS: Luminex platform
Sample: 4 x 3.2mm discs
LLD 1
LLD:
1-5
5 pg/mL
/ L
Reliability: Not great
Multiplex analysis of inflammatory
cytokines in DBS: MesoScale Discovery

Electrochemiluminescent
immunoassay

Microtiter plate-based
(
(96-well)
)

Quantify up to 10 biomarkers
Multiplex assay of cytokines in DBS
Protocol highlights
Assay performance
Adv/disadv

Platform: MSD Pro-Inflammatory 7-plex, Ultrasensitive

Calibrators: DBS material manufactured in lab with
serum calibrators provided in kit

Sample quantity: 2 x 3/16 in discs per well
 4 discs for duplicate measures (1 large drop)

Samples and calibrators eluted in filter plates to
maximize recovery
Multiplex assay of cytokines in DBS
Multiplex assay of cytokines in DBS
Multiplex assay of cytokines in DBS
Protocol highlights
Assay performance
Adv/disadv
IL6
IL10
TNFα
Lower limits of detection (pg
(pg/mL))
0.15
0.36
0.70
Linearity of dilution (mean)
1.03
1.03
1.04
Intra-assay variability (%CV)
5.6
4.0
7.5
Inter-assay variability (%CV)
-- tbd --
Multiplex assay of cytokines in DBS
Assay performance
Protocol highlights
Adv/disadv
Scatter Plot with Passing & Bablok Fit
N=72
25
Plasma IL6
6 (pg/mL)
IL6
30
20
15
10
Plasma =1.80 x DBS – 0.86
R = 0.98
5
0
0
5
10
DBS IL6 (pg/mL)
15
20
Multiplex assay of cytokines in DBS
Assay performance
Protocol highlights
Adv/disadv
Scatter Plot with Passing & Bablok Fit
4.5
IL6
4
N=70
Plasma IL6
6 (pg/mL)
3.5
3
2.5
2
1.5
Plasma =1.84 x DBS – 0.88
R = 0.71
1
0.5
0
0
0.5
1
1.5
2
DBS IL6 (pg/mL)
2.5
3
Multiplex assay of cytokines in DBS
Assay performance
Protocol highlights
Adv/disadv
Scatter Plot with Passing & Bablok Fit
N=71
10
R&D plasm
ma (pg/mL)
IL6
12
8
6
4
Plasma =1.95 x DBS – 0.88
R = 0.89
2
0
0
1
2
3
4
DBS IL6 (pg/mL)
5
6
7
Multiplex assay of cytokines in DBS
Assay performance
Protocol highlights
Adv/disadv
Scatter Plot with Passing & Bablok Fit
N=72
25
Plasma IL1
10 (pg/mL)
IL10
30
20
15
10
Plasma = 4.94 x DBS – 2.13
R = 0.71
5
0
0
2
4
6
8
DBS IL10 (pg/mL)
10
12
Multiplex assay of cytokines in DBS
Protocol highlights
Assay performance
Adv/disadv
Advantages

Tool for measuring several cytokines at very low
concentrations

Wide
de dy
dynamic
a c range:
a ge <0.5
0 5 pg/
pg/mL to
o 5,000 pg/
pg/mL

Reduced cost per biomarker/per sample
Disadvantages

Requires 1-2 drops of whole blood (for duplicates)

Big up
up-front
front instrumentation costs
Acknowledgements
Funding
g
NIA Network on Biological Risk (R24AG037898)
USC/UCLA Center on Biodemography and Population
Health (P30AG017265)
National Science Foundation, Physical Anthropology
(BCS-1027687)
Colleagues in the Laboratory for Human Biology Research
Elizabeth Miller
Bill Funk
Adam Leigh
American Journal of Human Biology
Impact factor:
Indexed:
Time to decision:
2.3
Everywhere
27 days
Toolkit: Methods in Human Biology
• Laboratory and field methods for
measuring human energy expenditure
• Field
Fi ld and
d llaboratory
b t
methods
th d iin
human milk research
• Essential anthropometric methods

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